We have performed a comprehensive quantitative proteomic study to analyze aortic proteins from young (8 mo) and old (30 mo) FXBN rats. Using 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE), we obtained 2-D gel maps of 301 identified non-redundant proteins from rat aorta and observed 18 proteins whose abundance significantly changes with aging. Utilizing Isobaric tags for relative and absolute quantitation (iTRAQ), 921 proteins were quantified and between both methods, 50 proteins were shown to have significantly different age-associated abundance. Proteomic analysis shows that one protein of interest, milk fat globule protein E8 (MFG-E8), significantly increases in abundance in old rat aortae. Transcription and translation analysis demonstrated that aortic MFG-E8 mRNA and protein levels increase with aging in several mammalian species, including humans. Dual immunolabeling shows that MFG-E8 colocalizes with both angiotensin II (Ang II) and monocyte chemoattractant protein-1 (MCP-1) within rat vascular smooth muscle cells (VSMCs) of the thickened aged aortic wall. AngII treatment of early passage VMSCs, isolated from young aortae, markedly increases MFG-E8 expression and enhances the invasive capacity of VSMCs to levels observed from old. MFG-E8 treatment of VSMCs increased MCP-1 and VSMC invasion capacity but these effects were inhibited by the MCP-1 receptor blocker, vCCI. Silencing MFG-E8 RNA substantially reduced MFG-E8 expression and VSMCs invasion capacity. Thus, arterial MFG-E8 significantly increases with aging and is a key component within the Ang II-MCP-1/VSMCs invasion signaling cascade. We have identified that aging arterial MFG-E8-enriched VSMCs are activated and proliferating both in vivo and in vitro. Increased MFG-E8 in VSMCs triggers phosphorylation of extracellular signal regulated kinases-1/2(ERK-1/2), augments levels of proliferation cell nuclear antigen (PCNA) and cyclin-dependent kinase 4 (CDK4), increases BrdU incorporation and promotes cell growth. When MFG-E8 is knocked down the speed of cell cycling is reduced, the expression of the signaling molecules PCNA and CDK4 is accelerated, thus facilitating the cells entry into a growth-arrested state. Furthermore, we find that integrin v5 and platelet derived growth factor (PDGF) receptor are upregulated with MFG-E8 treatment and are also key elements of proliferative signals in aging VSMC. In addition, MFG-E8 facilitates the profibrosis of VSMCs with aging via an interaction with caveolin 1 (CAV1) signaling. Exposure of isolated aortic VSMCs from old rats to MFG-E8 increases the profibrogenic signaling molecules TGF-1, TGF- receptor type II (TRII), p-SMAD (Sma and Mad: Mothers against decapentaplegic)2/3, and collagen I (Col I) while knockdown of MFG-E8 gene expression substantially reduces the expression of these molecules. Both co-immuno-labeling and co-immune-precipitation of MFG-E8, TRII, and CAV1 in young VSMC (8-mo) indicates their protein-protein physical association. In young VSMCs, MFG-E8 up-regulates the expression of CAV1. Knocking down the CAV1 gene, like MFG-E8 exposure, significantly increases the expression of TGF-1, TRII, p-SMAD-2/3, and Col I. Similarly, overexpressing the CAV1 gene, like MFG-E8 gene knockdown, markedly decreases the expression of these fibrogenic molecules in both young and old VSMCs. Furthermore, MFG-E8 treatment significantly increases the expression of TGF-1, p-SMAD-2/3, and Col I in CAV1 silenced young VSMC. Interestingly, MFG-E8 exposure does not increase expression of these profibrogenic molecules in old VSMCs when CAV1 is overexpressed. These results, for the first time, demonstrate that MFG-E8 modulates the TGF-1 fibrogenic signaling in VSMC in a CAV1/age-dependent manner. Importantly, we found that the involvement of MFG-E8 in atherosclerotic lesions increases with age. In in vivo studies: 1) Immunostaining demonstrates that MFG-E8 protein in the aortic wall of ApoE-/- mice on a standard chow diet increases with age and is localized predominantly in advanced plaques and elastin laminae. 2) In nonhuman primates (9 to 23- years old), the iTRAQ assay indicates that levels of arterial MFG-E8 protein is highly correlated with age. In older monkeys (>17-years old), fed a high cholesterol diet for 2 years, a 1.6-fold increase in the amount of arterial MFG-E8 protein and its fragment, medin, was seen when compared to older animals fed a standard diet. 3) In aortic thoracic intimae isolated from adult humans (age 22-to 69-years old), Western blot analysis indicated that the abundance of aortic MFG-E8 protein and its fragment, medin, significantly increased with advancing age; 4) Immunostaining of atherosclerotic plaques indicates that MFG-E8 is mainly derived from smooth muscle cells (alpha-smooth actin positive) and macrophages (CD 68 positive). In in vitro studies: 1) Early passage thoracic aortic smooth muscle cells (SMC) from both young and old monkeys treated with pathogenic oxidative low-density lipoprotein (oxLDL) showed an increase in the uptake capacity of oxLDL in old cells that exceeded that of young cells, thus facilitating the transdifferentiation of SMCs into foam-like cells (CD68 positive). 2) Silencing of MFG-E8 RNA markedly reduced the uptake of oxLDL by young and old monkey SMC. It is known that aging increases MFG-E8 expression in the rat, nonhuman primate and human aortic walls, facilitating the invasion, proliferation, and proinflammation of VSMCs. The MFG-E8 C-terminal fragment, medin, was reported to be involved in the necrosis of VSMCs at the inflammatory sites of aortic dissection. Our recent immunofluorescence, immunohistochemistry and western blot analysis demonstrate that MFG-E8 and its fragment, medin, activated matrix metalloproteinase type II (MMP-2) and platelet derived growth factor receptor type-beta (PDGFR-) protein levels were markedly increased in human grossly normal aortic walls from old (n=10, >50 yrs) vs. younger donors (n=10, <20 yrs). Importantly, treating primary cultured VSMCs isolated from young (8 mo) and old (30 mo) FXBN rat aortae to a peptide of medin (20 to100nM) significantly increased MMP-2 activation, PDGFR- expression, and migratory capacity measured with a modified Boyden chamber in a dose-dependent manner. Exposure of activated MMP-2 to recombinant human MFG-E8 protein or to old human aortic protein markedly increased the cleavage product medin, respectively and both were substantially inhibited by GM6001, an MMP inhibitor. Treating VSMCs with medin did not significantly affect the expression of cell cycle related proteins, including proliferating cellular nuclear antigen (PCNA), and the proliferation of VSMC as determined by a cell proliferation MTT assay. In addition, platelet-derived growth factor BB (PDGF-BB) treatment markedly activated MMP-2 in both young and old VSMCs, which was substantially reduced by a PDGFR- inhibitor, RTK. Taken together, in vivo and in vitro observations from human donors and rat specimens demonstrate that aging increases aortic medin protein levels, via an MMP-2 cleavage of MFG-E8, promoting VSMCs inflammation and migration, a feature of aged aortic walls. Thus, targeting MFG-E8 or its cleavage product medin is a novel approach to the prevention or treatment of large arterial aging or age-associated disease.